This project is exploring the structure and function of important phagocytic cell proteins. Current efforts are focused on the components of the NADPH oxidase, an important host defense system that produces superoxide anion and related microbicidal oxidants. The cDNAs encoding all five known oxidase components have been expressed in several recombinant DNA expression systems (bacterial, insect and mammalian) enabling systematic manipulation of their structures and identification of relevant functional domains. Oxidase activity was restored to B-cells from patients with chronic granulomatous disease (CGD) by transfection with episomal expression vectors. Other more efficiently transfected model cell lines were also explored, including K562 cells, which produced superoxide when transfected with all four CGD genes together. Structural features critical to oxidase regulation by the fifth, ras-related G- protein, oxidase component (rac) have also been explored by comparing activities of chimeric recombinant proteins which have incorporated sequences from inactive ras homologs. The role of conserved src homology domains (SH3), found in p47-phox and p67-phox, on oxidase assembly and activation has been studied extensively. Comparison of the effects of deletions of these domains in both cell-free and intact transfected cell assays indicated a critical role in the membrane translocation of these proteins. Three proline-rich sites which bind to the src homology domains were identified within p47-phox and the small subunit of the transmembrane cytochrome b558 (p22-phox). A single proline mutation within p22-phox, observed in one form of CGD, was shown to abolish binding to the src homology domains of p47-phox. Competitive peptide binding studies suggest that switching from intramolecular to intermolecular interactions with the src-like domains in p47-phox enables its association with the membrane bound cytochrome during translocation. The target sites for SH3 domains in the oxidase system conform well with a consensus of other SH3 binding sequences defined in other systems (abl and GRB2, a mediator of EGF to ras signalling). Thus, these findings provide insights on a variety of signal transduction cascades which utilize this same conserved protein motif. These studies may aid in the design of agents which modulate the activity of this important inflammatory process and may facilitate development of gene therapy for CGD.